In-vitro intestinal activity maintaining device and application thereof

Abstract

The invention discloses an in-vitro intestinal activity maintaining device and application thereof. The temperature of the liquid storage tank and the temperature control water tank is controlled by a temperature control device; a temperature control chamber and a culture cavity are arranged in the isolated double tank, a multi-outlet liquid and gas pipeline is arranged at the bottom of the culture cavity, three liquid inlet pipelines, three liquid outlet pipelines and a gas inlet pipeline are arranged in the temperature control chamber, and the three liquid inlet pipelines and the three liquid outlet pipelines are respectively used for filling nutrient solution into the cavity, mesenteric blood vessels and the inner tank; the temperature control water tank is communicated with the temperature control chamber, the gas source is communicated with the multi-outlet gas pipeline through the gas inlet pipeline, the gas source is communicated with the liquid storage tank, the liquid storage tank is respectively communicated with the three liquid inlet pipelines, and the waste liquid storage tank is respectively communicated with the three liquid outlet pipelines. According to the device, the oxygenation capacity and the oxygen supply capacity are improved through two-way air-blowing oxygenation, multiple air-blowing sites and multi-channel perfusion, the temperature control stability is improved through preheating or precooling, and the activity of a thick tissue sample can be effectively kept.

Description

Technical Field

[0001] The present invention belongs to the field of medical equipment, and in particular relates to an isolated intestinal activity maintaining device and application thereof. Background Art

[0002] The ability to maintain and monitor living intestinal tissue in vitro is of great value for scientific research and clinical translation. In biomedical research, isolated intestinal models provide a highly controllable platform, enabling researchers to delve into the complex functions of the gastrointestinal tract, such as motility, secretion, nutrient absorption, and barrier function, under reproducible experimental conditions. Compared to monolayer cell cultures, in vitro intestinal tissue more fully retains the native multicellular architecture, including connective tissue cells, enteric neurons, immune cells, and the epithelial cells that make up the intestinal wall, thus more realistically mimicking the in vivo microenvironment. The construction of physiologically active in vitro intestinal models allows researchers to systematically study key biological processes such as drug absorption mechanisms, nutrient sensing signaling, and host-microbe interactions in an environment that more closely resembles in vivo physiological conditions. In terms of clinical applications, the maintenance of isolated intestinal viability and related technologies show broad promise in drug development, disease modeling, and organ transplantation. Therefore, the development of efficient systems for maintaining isolated intestinal viability will not only facilitate in-depth research on gastrointestinal physiological mechanisms but also promote advances in clinical applications, such as improving the reliability of drug safety assessments, optimizing transplantation strategies, and accelerating the implementation of personalized medicine programs based on patient-derived tissue.

[0003] Maintaining the viability of isolated intestinal tissue requires reproducing the key physiological conditions experienced in vivo. Key factors for all methods include oxygenation, nutrient delivery, waste removal, and temperature control, all of which are designed to prevent ischemia and cell death in vitro. The intense metabolic activity of intestinal tissue requires a continuous oxygen supply. Thinner intestinal tissues (such as those of rats) can often rely on diffusion from an oxygenated bath. For example, current methods involve immersing intestinal segments in Kerbs-buffered physiological solution and bubbling with 95% O₂ / 5% CO₂ to promote oxygen diffusion into the tissue. However, larger intestinal segments (such as those of porcine or human) are much thicker, and immersion in a single bubbling medium rapidly leads to the formation of an oxygen-deficient core. Therefore, for thicker intestinal tissue, in addition to the aforementioned controlled conditions, conditions that promote oxygenation, diffusion, and perfusion should be established. Furthermore, in addition to studying intestinal physiology at body temperature, the physiological activity and metabolism of intestinal tissue in cryopreservation solutions are also of interest. Therefore, viability preservation systems also need to provide cryocontrol while maintaining oxygenation, nutrient delivery, and waste removal. However, the existing method only uses a single bubbling method and the diffusion effect of oxygen-containing bath fluid, which is only applicable to the activity maintenance of thin tissues such as mouse intestines. It does not consider the problem of oxygen overflow from the open liquid surface and the control conditions for prolonging activity maintenance such as low temperature. Summary of the Invention

[0004] To address the shortcomings of existing technologies and meet practical needs, the present invention provides an in vitro intestinal activity maintenance device and its application. This device improves oxygenation and supply capacity through dual-pass aeration oxygenation, multiple aeration sites, and multi-channel perfusion. It also enhances temperature control stability through preheating or precooling, effectively maintaining the activity of thick tissue samples.

[0005] The present invention adopts the following technical solutions:

[0006] 1. An in vitro intestinal activity maintenance device

[0007] The in vitro intestinal activity maintenance device includes an isolated double tank, a liquid storage tank, a temperature-controlled water tank, an air source, a temperature control device and a waste liquid storage tank; the temperature of the nutrient solution in the liquid storage tank and the temperature-controlled water in the temperature-controlled water tank are both controlled by the temperature control device; a temperature control chamber and a culture cavity are provided in the isolated double tank, the culture cavity is located in the temperature control chamber, and a multi-outlet liquid pipeline and a multi-outlet gas pipeline are arranged at the bottom of the culture cavity, the culture cavity is used to place in vitro intestinal samples, and three liquid inlet pipelines, three liquid outlet pipelines and one air inlet pipeline are arranged in the temperature control chamber, and the three liquid inlet pipelines and the liquid outlet pipelines correspond to each other one by one and are used to perfuse nutrient solution into the cavity, mesenteric blood vessels and inner tank respectively, so as to realize multi-channel tissue fluid perfusion; the temperature-controlled water tank It is interconnected with the temperature control chamber through a temperature-controlled water circulation pipeline, the gas source is connected to the inlet of the air inlet pipeline, the outlet of the air inlet pipeline is connected to the inlet of the multi-outlet gas pipeline, the gas source is connected to the air inlet of the liquid storage tank, the liquid outlet of the liquid storage tank is connected to the inlets of the three liquid inlet pipelines respectively, the outlet of the inner tank perfusion liquid inlet pipeline is connected to the inlet of the multi-outlet liquid pipeline, the outlets of the intracavitary perfusion and mesenteric vascular perfusion liquid inlet pipelines are respectively used to fix the first end of the intestine and mesenteric blood vessels of the isolated intestinal sample, the inlets of the intracavitary perfusion and mesenteric blood vessel perfusion liquid outlet pipelines are respectively used to fix the second end of the intestine and mesenteric blood vessels of the isolated intestinal sample, and the inlet of the waste liquid storage tank is connected to the outlets of the three liquid outlet pipelines respectively.

[0008] The isolated intestinal activity maintaining device further comprises:

[0009] A buffer bottle is installed on at least one of the liquid inlet pipeline and / or the air inlet pipeline; the air and liquid are allowed to fully exchange energy with the external circulation environment to ensure that they have a controllable temperature before entering the intestinal tissue;

[0010] A check valve is installed on at least one of the liquid inlet pipeline and / or the air inlet pipeline to prevent the nutrient solution and oxygen-containing gas from flowing back after the flow path in the device stops circulating;

[0011] Solution pump: A solution pump is installed between the liquid outlet of the liquid storage tank and the inlet of each liquid inlet pipeline, and a solution pump is installed on the temperature-controlled water circulation pipeline as an external circulation water pump;

[0012] Gas mass flow meters are installed on the pipelines between the gas source and the liquid storage tank, and between the gas source and the air inlet pipeline.

[0013] Specifically, the isolated double tank is mainly composed of an outer tank shell, an inner tank shell, a cover plate and a silicone rubber base. The inner tank shell and the outer tank shell both adopt an open top structure. The inner tank shell is arranged inside the outer tank shell. The middle area of the outer tank shell and the inner tank shell serves as a temperature control chamber. A cover plate is arranged above the temperature control chamber. The cover plate is detachably and sealedly connected to the outer tank shell and the inner tank shell to ensure that the temperature-controlled water in the interlayer circulates under the action of water pressure; the cavity inside the inner tank shell is a culture chamber, and the silicone rubber base is laid at the bottom of the culture chamber. Each branch outlet of the multi-outlet liquid pipeline and the multi-outlet gas pipeline extends vertically upward to the top surface of the silicone rubber base and opens upward, so that the nutrient solution and oxygen-containing gas are passed into the culture chamber from bottom to top.

[0014] Specifically, the isolated intestinal sample is an isolated intestinal sample from a pig or a human, the isolated intestinal sample includes the intestine and / or mesenteric blood vessels, and the intestine is an intestinal segment or an intestinal slice.

[0015] Furthermore, the outer tank shell of the isolated double tank is provided with a plurality of outer tank liquid inlet holes, outer air inlet holes and outer tank liquid outlet holes, and the inner tank shell is provided with a plurality of inner tank liquid inlet holes, inner air inlet holes and inner tank liquid outlet holes. The outer tank liquid inlet holes are connected to the corresponding inner tank liquid inlet holes through pipelines, the outer tank liquid outlet holes are connected to the corresponding inner tank liquid outlet holes through pipelines, and the outer air inlet holes are connected to the corresponding inner air inlet holes through pipelines, forming liquid inlet pipelines, liquid outlet pipelines and air inlet pipelines; the holes all have an internal thread structure and are connected to the pipelines through threaded connections.

[0016] Furthermore, the isolated double tank also includes an external circulation water inlet and an external circulation water outlet; the external circulation water inlet is opened at the upper part of the outer tank shell, and is connected to the outlet of the temperature-controlled water tank through a pipeline, and the external circulation water outlet is opened at the lower part of the outer tank shell, and is connected to the inlet of the temperature-controlled water tank through a pipeline; the temperature-controlled water tank is placed on the temperature control platform of the temperature control device, and a liquid storage tank is placed in the temperature-controlled water tank; the liquid inlet pipeline, liquid outlet pipeline and air inlet pipeline are all immersed in the temperature-controlled water in the temperature-controlled chamber, so that the nutrient solution and oxygen-containing gas are preheated or precooled by the temperature-controlled water before entering the culture chamber and the in vitro intestinal sample.

[0017] Furthermore, the isolated double tank also includes an intracavity perfusion inner tank liquid inlet hole, an intracavity perfusion inner tank liquid outlet hole, a mesenteric vascular perfusion inner tank liquid inlet hole and a mesenteric vascular perfusion inner tank liquid outlet hole opened on the inner tank shell; the intracavity perfusion inner tank liquid inlet hole serves as the outlet of the intracavity perfusion liquid inlet pipeline, the intracavity perfusion inner tank liquid outlet hole serves as the inlet of the intracavity perfusion liquid outlet pipeline, the mesenteric vascular perfusion inner tank liquid inlet hole serves as the outlet of the mesenteric vascular perfusion liquid inlet pipeline, and the mesenteric vascular perfusion inner tank liquid outlet hole serves as the inlet of the mesenteric vascular perfusion liquid outlet pipeline; with the length direction of the isolated intestinal sample as the first direction, the intracavity perfusion inner tank liquid inlet hole and the intracavity perfusion inner tank liquid outlet hole are arranged relative to each other in the first direction, and the line connecting the two is parallel to the first direction; the mesenteric vascular perfusion inner tank liquid inlet hole and the mesenteric vascular perfusion inner tank liquid outlet hole are arranged relative to each other in the first direction, and the line connecting the two is parallel to the first direction.

[0018] Furthermore, the isolated double trough also includes an intracavitary perfusion outer trough inlet, an intracavitary perfusion outer trough outlet, a mesenteric vascular perfusion outer trough inlet, and a mesenteric vascular perfusion outer trough outlet, which are provided on the outer trough shell. The intracavitary perfusion outer trough inlet and the intracavitary perfusion inner trough inlet are connected by a pipeline to form an intracavitary perfusion inlet pipeline. The intracavitary perfusion inner trough outlet and the intracavitary perfusion outer trough outlet are connected by a pipeline to form an intracavitary perfusion outlet pipeline. The mesenteric vascular perfusion outer trough inlet and the mesenteric vascular perfusion inner trough inlet are connected by a pipeline to form a mesenteric vascular perfusion inlet pipeline. The mesenteric vascular perfusion inner trough outlet and the mesenteric vascular perfusion outer trough outlet are connected by a pipeline to form a mesenteric vascular perfusion outlet pipeline. The inlet of the intracavitary perfusion inlet pipeline is the intracavitary perfusion outer trough inlet, and the outlet is the intracavitary perfusion inner trough inlet. The inlet of the intraluminal perfusion outlet line is the intraluminal perfusion inner tank outlet hole, and the outlet is the intraluminal perfusion outer tank outlet hole. The inlet of the mesenteric vascular perfusion inlet line is the mesenteric vascular perfusion outer tank inlet hole, and the outlet is the mesenteric vascular perfusion inner tank inlet hole. The inlet of the mesenteric vascular perfusion outlet line is the mesenteric vascular perfusion inner tank outlet hole, and the outlet is the mesenteric vascular perfusion outer tank outlet hole.

[0019] Furthermore, the isolated double tank also includes an outer inlet for nutrient solution perfusion in the inner tank and an outer inlet for nutrient solution perfusion in the inner tank, as well as an inner inlet and inner outlet for nutrient solution perfusion in the inner tank, also provided on the inner tank shell. The outer inlet for nutrient solution perfusion in the inner tank and the inner inlet for nutrient solution perfusion in the inner tank are connected by a pipeline to form an inner tank perfusion inlet pipeline. The inlet of the inner tank perfusion inlet pipeline is the outer inlet for nutrient solution perfusion in the inner tank, and the outlet is the inner inlet for nutrient solution perfusion in the inner tank. The inner outlet for nutrient solution perfusion in the inner tank and the outer outlet for nutrient solution perfusion in the inner tank are connected by a pipeline to form an inner tank perfusion outlet pipeline. The inlet of the inner tank perfusion outlet pipeline is the inner outlet for nutrient solution perfusion in the inner tank, and the outlet is the outer outlet for nutrient solution perfusion in the inner tank. The inner inlet for nutrient solution perfusion in the inner tank is located at the bottom of the inner tank shell and is connected to the inlet of the multi-outlet liquid pipeline. The inner outlet hole for the nutrient solution injection in the inner tank is located at the upper part of the inner tank shell.

[0020] Furthermore, the isolated double tank also includes an external air inlet opening formed in the outer tank shell and an internal air inlet opening formed in the inner tank shell. The external and internal air inlet openings are connected by a pipeline to form an air inlet pipeline. The inlet pipeline has an external air inlet opening as its inlet and an internal air inlet opening as its outlet. The internal air inlet opening is located at the bottom of the inner tank shell and is connected to the inlet of the multi-outlet gas pipeline.

[0021] During the process of solution circulation and introduction of oxygen-containing gas, the nutrient solution and oxygen-containing gas flow through the interlayer before being transported into the inner tank structure and the isolated intestinal sample, and are preheated or precooled under the regulation of the temperature-controlled circulating water temperature in the interlayer.

[0022] Furthermore, the isolated intestinal activity maintaining device further comprises a venous effluent storage tank, and the mesenteric vascular perfusion effluent pipeline is connected to the venous effluent storage tank.

[0023] 2. Application of an in vitro intestinal activity maintenance device

[0024] Used for activity maintenance and physiological activity testing of ex vivo intestinal samples, the physiological activity testing including electrophysiological testing, muscle tension testing, microscopic observation under active conditions, controlled pressure perfusion and intraluminal pressure testing, the ex vivo intestinal sample is an ex vivo intestinal sample from a pig or a human, the ex vivo intestinal sample includes the intestine and / or mesenteric blood vessels, and the intestine is an intestinal segment or intestinal slice.

[0025] 3. Application Method of an In Vitro Intestinal Activity Preservation Device

[0026] The application method comprises the following steps:

[0027] S1) turning on the temperature control device and the external circulation water pump to make the temperatures of the nutrient solution in the liquid storage tank and the temperature-controlled water in the temperature-controlled water tank reach a preset temperature;

[0028] S2) turning on the gas source to deliver oxygen-containing gas to the liquid storage tank and the culture chamber of the isolated double tank respectively;

[0029] S3) perfusing the culture chamber with oxygenated nutrient solution through the inner tank perfusion inlet and outlet pipes;

[0030] S4) If the intestinal tract of the isolated intestinal sample is an intestinal segment, connecting and securing both ends of the intestinal tract to the inlet and outlet of the intraluminal perfusion tank, respectively, and perfusing the intestinal tract with oxygenated nutrient solution through the intraluminal perfusion inlet and outlet pipes;

[0031] If the intestinal fragment of the isolated intestinal sample is an intestinal fragment, the intestinal fragment is fixed to the bottom of the culture chamber using a sample fixing needle, and oxygenated nutrient solution is perfused into the bottom of the intestinal fragment through the intraluminal perfusion inlet and outlet pipes;

[0032] S5) connecting and fixing the two ends of the mesenteric blood vessels of the isolated intestinal sample to the inlet and outlet holes of the mesenteric blood vessel perfusion inner tank, respectively, and perfusing the mesenteric blood vessels with oxygenated nutrient solution through the mesenteric blood vessel perfusion inlet and outlet pipes;

[0033] S6) Preserving the ex vivo intestinal sample at a preset storage temperature or incubating it at a preset test temperature for a period of time, and then performing a physiological activity test.

[0034] The step S6 specifically includes: storing the isolated intestinal sample at a temperature of 3 to 5° C. or incubating it at a temperature of 36 to 38° C. for 15 to 60 minutes, and then performing a physiological activity test.

[0035] The physiological activity test is an electrophysiological test, a muscle tension test, a microscopic observation under active conditions, or a controlled pressure perfusion and intracavitary pressure test;

[0036] The electrophysiological test process specifically includes: extracting part of the intestinal wall tissue to the end of the glass tube suction electrode by negative pressure, connecting the glass tube suction electrode to the electrophysiological test system and the host computer, and collecting the electrophysiological test signal;

[0037] The muscle tension test process is specifically as follows: fixing one end of the intestine to the bottom of the inner tank, connecting the other end to a force sensor, electrically connecting the force sensor to a host computer, and collecting muscle tension test signals;

[0038] The microscopic observation process is specifically as follows: a light source is integrated below the isolated intestinal sample, a microscope is arranged above the sample, and the microscopic structure of the isolated intestinal sample is observed using the microscope;

[0039] The controlled pressure perfusion and intracavitary pressure testing process is specifically as follows: connecting the intracavitary perfusion outlet pipeline and / or the mesenteric vascular perfusion outlet pipeline to an electronic water pressure gauge, connecting the force sensor to the upper computer, and monitoring the pressure changes in the cavity and / or mesenteric blood vessels in real time.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] 1. To meet the need for maintaining the activity of thick tissues such as the human intestine, the present invention proposes an in vitro intestinal activity maintenance device based on an isolated double-tank structure. This device performs dual-pass aeration oxygenation on the liquid surface of the open tissue tank and the liquid storage system, and provides multiple aeration points in the tissue tank, systematically improving the oxygenation capacity of the device and providing an environmental foundation for maintaining the activity of thick tissues.

[0042] 2. The present invention utilizes the temperature environment of the outer tank to preheat or precool the perfusion solution, thereby improving the stability of the device in controlling the tissue environment temperature;

[0043] 3. The present invention sets up a multi-channel tissue fluid perfusion system. In the case of mesenteric blood vessels after intestinal resection, multi-channel perfusion is performed outside the intestine, inside the intestine, and inside the blood vessels, thereby comprehensively improving the oxygen supply capacity, preventing the formation of hypoxic cores and necrosis inside thick tissues, and prolonging the maintenance time of active intestine.

[0044] 4. In addition to providing temperature control capabilities for simulating body temperature conditions and low-temperature storage conditions, the device also adds low-temperature or sub-normal temperature control capabilities, providing a basis for physiological testing under multiple environmental conditions;

[0045] 5. The present invention also provides an application method of the isolated intestinal activity maintenance device in electrophysiological testing, muscle tension testing, microscopic imaging and intracavitary pressure testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Schematic diagram of the three-dimensional structure of the isolated double tanks in the in vitro intestinal activity maintenance device of the present invention;

[0047] Figure 2 Schematic cross-sectional view of the isolated double tanks in the in vitro intestinal activity maintenance device of the present invention;

[0048] Figure 3 This is an overall schematic diagram of the in vitro intestinal activity maintenance device of the present invention;

[0049] Figure 4 Schematic diagram of the application of the isolated intestinal activity maintenance device of the present invention in intestinal electrophysiological testing;

[0050] Figure 5 Schematic diagram of the application of the isolated intestinal activity maintenance device of the present invention in muscle tension testing;

[0051] Figure 6 Schematic diagram of the application of the in vitro intestinal activity maintenance device of the present invention in microscopic testing;

[0052] Figure 7 Schematic diagram of the application of the isolated intestinal activity maintenance device of the present invention in controlled pressure perfusion and intraluminal pressure testing;

[0053] Figure 8 Schematic diagram of the air supply and nutrient solution supply of a conventional in vitro intestinal activity maintenance device in a comparative example of the present invention.

[0054] In the figure, 1, outer tank shell, 2, inner tank shell, 3, cover plate, 4-1, external circulation water inlet, 4-2, external circulation water outlet, 5-1, outer tank inlet for intracavitary perfusion, 5-2, inner tank inlet for intracavitary perfusion, 5-3, inner tank outlet for intracavitary perfusion, 5-4, outer tank outlet for intracavitary perfusion, 6-1, outer tank inlet for mesenteric vascular perfusion, 6-2, inner tank inlet for mesenteric vascular perfusion, 6-3, inner tank outlet for mesenteric vascular perfusion, 6-4, outer tank outlet for mesenteric vascular perfusion, 7-1, outer inlet for nutrient solution perfusion in inner tank, 7-2, inner inlet for nutrient solution perfusion in inner tank, 7-3, multi-outlet liquid pipeline, 8-1, outer air inlet, 8- 2. Internal air inlet, 8-3. Multi-outlet gas pipeline, 9-1. Internal outlet for nutrient solution perfusion in the inner tank, 9-2. External outlet for nutrient solution perfusion in the inner tank, 10. Silicone rubber base, 002. Second grating, 12. Mesenteric blood vessels, 13. Buffer bottle, 14. Check valve, 15. Gas source, 16. Gas mass flow meter, 17. Temperature control device, 18. Liquid storage tank, 19. Solution pump, 20. Waste liquid storage tank, 21. Glass tube suction electrode, 22. Electrophysiological test system, 23. Host computer, 24. Sample fixing needle, 25. Force sensor, 26. Microscope, 27. Light source, 28. Liquid pressure sensor, 29. Air inlet, 30. Liquid inlet. DETAILED DESCRIPTION

[0055] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0056] The first aspect of the present invention provides an in vitro intestinal activity maintenance device. The in vitro intestinal activity maintenance device of the present invention is aimed at larger and thicker intestinal samples such as pig or human intestinal segments. The liquid surface of the open tissue tank and the liquid storage system are subjected to double-pass aeration oxygenation, and multiple aeration sites are set for the tissue tank to systematically improve the oxygenation capacity of the device and provide an environmental basis for maintaining the activity of thick tissues; the temperature environment of the temperature control chamber in the outer tank is used to preheat or precool the perfusion fluid (oxygen-containing nutrient solution) to improve the stability of the device in controlling the temperature of the tissue environment; a multi-channel tissue fluid perfusion system is set up, and in the case of mesenteric blood vessels after human intestinal surgical resection, multi-channel perfusion is performed on the outside, inside and inside the intestinal tube, thereby comprehensively improving the oxygen supply capacity, preventing the formation of hypoxic cores and necrosis inside thick tissues, and prolonging the maintenance time of active intestines; in addition to temperature control under body temperature conditions, the device also adds low temperature or subnormal temperature control capabilities, providing a basis for physiological testing under multiple environmental conditions.

[0057] The present invention provides an isolated intestinal activity maintenance device comprising an isolated double tank, a liquid storage tank 18, a temperature-controlled water tank, a gas source 15, a temperature control device 17, and a waste liquid storage tank 20. Gas source 15 stores oxygen-containing gas, liquid storage tank 18 stores nutrient solution, and the temperature-controlled water tank stores temperature-controlled water. The temperatures of the nutrient solution in liquid storage tank 18 and the temperature-controlled water in the temperature-controlled water tank are both controlled by temperature control device 17. A temperature-controlled chamber and a culture cavity are provided within the isolated double tank. The culture cavity is located within the temperature-controlled chamber. A multi-outlet liquid pipeline 7-3 and a multi-outlet gas pipeline 8-3 are arranged at the bottom of the culture cavity. The culture cavity is used to accommodate isolated intestinal samples. The temperature-controlled chamber is provided with three liquid inlet pipelines, three liquid outlet pipelines, and one gas inlet pipeline. The three liquid inlet pipelines and the liquid outlet pipelines correspond to each other and are used to perfuse oxygen-containing nutrient solution into the cavity, mesenteric vessels, and inner tank, respectively.

[0058] The gas and liquid flow paths in the isolated intestinal activity maintenance device of the present invention are specifically connected as follows:

[0059] Temperature-controlled water circulation system: The temperature-controlled water tank and the temperature-controlled room are connected to each other through a temperature-controlled water circulation pipeline;

[0060] A dual-pass aeration oxygenation system with multiple aeration points: the gas source 15 is connected to the inlet of the gas inlet pipeline, the outlet of the gas inlet pipeline is connected to the inlet of the multi-outlet gas pipeline 8-3, and the gas source 15 is connected to the gas inlet of the liquid storage tank 18;

[0061] Multi-channel tissue fluid perfusion system: the liquid outlet of the liquid storage tank 18 is connected to the inlets of the three liquid inlet pipelines respectively, the outlet of the inner tank perfusion inlet pipeline for perfusing oxygen-containing nutrient solution into the inner tank is connected to the inlet of the multi-outlet liquid pipeline 7-3, the intraluminal perfusion inlet pipeline and the mesenteric vascular perfusion inlet pipeline for perfusing oxygen-containing nutrient solution into the cavity and mesenteric blood vessels, the outlets of which are respectively used to fix the first ends of the intestine 11 and the mesenteric blood vessels 12 of the isolated intestinal sample, the inlets of the intraluminal perfusion outlet pipeline and the mesenteric blood vessels perfusion outlet pipeline for perfusing oxygen-containing nutrient solution into the cavity and mesenteric blood vessels are respectively used to fix the second ends of the intestine 11 and the mesenteric blood vessels 12 of the isolated intestinal sample, and the inlet of the waste liquid storage tank 20 is connected to the outlets of the three liquid outlet pipelines respectively.

[0062] Furthermore, the isolated intestinal activity maintaining device further comprises:

[0063] A buffer bottle 13 is installed on at least one liquid inlet pipeline and / or air inlet pipeline; the air and liquid are allowed to fully exchange energy with the external circulation environment to ensure that they have a controllable temperature before entering the intestinal tissue;

[0064] A check valve 14 is installed on at least one of the liquid inlet pipeline and / or the air inlet pipeline to prevent the oxygen-containing nutrient solution and the oxygen-containing gas from flowing back after the flow path in the device stops circulating;

[0065] Solution pump 19, a solution pump 19 is installed on the pipeline between the liquid outlet of the liquid storage tank 18 and the inlet of each liquid inlet pipeline, and a solution pump 19 is installed on the temperature-controlled water circulation pipeline as an external circulation water pump;

[0066] A gas mass flow meter 16 is installed on each of the pipelines between the gas source 15 and the liquid storage tank 18 and between the gas source 15 and the air intake pipeline.

[0067] Preferably, a buffer bottle 13 is installed on each of the inner tank perfusion liquid inlet pipeline and the air inlet pipeline. A check valve 14 is installed on each of the inner tank perfusion liquid inlet pipeline and the air inlet pipeline to prevent the oxygen-containing nutrient solution and the oxygen-containing gas from flowing back after the flow path in the device stops circulating.

[0068] Specifically, the isolated double tank is primarily composed of an outer tank shell 1, an inner tank shell 2, a cover plate 3, and a silicone rubber base 10. Both the inner tank shell 2 and the outer tank shell 1 have open-top structures. The inner tank shell 2 is arranged inside the outer tank shell 1. The middle area between the outer tank shell 1 and the inner tank shell 2 serves as a temperature control chamber. A cover plate 3 is arranged above the temperature control chamber. The cover plate 3 is removably and sealedly connected to the outer tank shell 1 and the inner tank shell 2 to ensure that the temperature-controlled water in the interlayer circulates under the action of water pressure. The cavity inside the inner tank shell 2 serves as the culture chamber. The silicone rubber base 10 is laid at the bottom of the culture chamber. Each branch outlet of the multi-outlet liquid pipeline 7-3 and the multi-outlet gas pipeline 8-3 extends vertically upward to the top surface of the silicone rubber base 10 and opens upward, allowing oxygen-containing nutrient solution and oxygen-containing gas to be introduced into the culture chamber from bottom to top.

[0069] Specifically, the inner tank housing 2 can be fixed by being sealed with the cover plate 3 .

[0070] Specifically, the isolated intestinal sample is an isolated intestinal sample from a pig or a human, and the isolated intestinal sample includes the intestine 11 and / or mesenteric blood vessels 12 , and the intestine 11 is an intestinal segment or an intestinal piece.

[0071] Specifically, the silicone rubber substrate 10 is formed on the bottom of the culture chamber by in-situ curing.

[0072] Preferably, the silicone rubber base 10 adopts a contoured design so that silicone rubber interfaces protrude at different positions of the entire intestine 11, so that the oxygen-containing nutrient solution and oxygen-containing gas bulging out of the outlets of the multi-outlet liquid pipeline 7-3 and the multi-outlet gas pipeline 8-3 can fully contact the intestine 11.

[0073] Preferably, the temperature-controlled water tank is placed on the temperature-controlled platform of the temperature control device 17 , and the liquid storage tank 18 is placed in the temperature-controlled water tank.

[0074] Preferably, the internal threaded holes are connected to the threaded hose connectors to form the liquid inlet, liquid outlet, and air inlet lines in the temperature control chamber, achieving complete sealing of the outer tank structure. This ensures that the rapidly flowing circulating temperature-controlled water in the outer shell does not affect the stability of the physiological test of the inner tank tissue. At the same time, by regulating the circulating water temperature, the overall stable control of the device temperature environment is achieved, improving the operability of controlling the in vitro intestinal physiological environment. Specifically: the outer tank shell 1 of the isolated double tank is provided with a plurality of outer tank liquid inlets, outer air inlets, and outer tank liquid outlets, and the inner tank shell 2 is provided with a plurality of inner tank liquid inlets, inner air inlets, and inner tank liquid outlets. The outer tank liquid inlets are connected to the corresponding inner tank liquid inlets via pipelines, the outer tank liquid outlets are connected to the corresponding inner tank liquid outlets via pipelines, and the outer air inlets are connected to the corresponding inner air inlets via pipelines, forming the liquid inlet, liquid outlet, and air inlet lines. These holes all have internal thread structures and are connected to the pipelines via threaded connections. For example, an intracavity perfusion outer tank liquid inlet hole 5-1 is opened on the outer tank shell 1, and the intracavity perfusion outer tank liquid inlet hole 5-1 is connected to one end of the hose through a thread, and the other end of the hose is connected to the intracavity perfusion inner tank liquid inlet hole 5-2 through a thread, forming an intracavity perfusion liquid inlet pipeline for perfusing oxygen-containing nutrient solution into the cavity.

[0075] Preferably, the isolated double tank also includes an external circulating water inlet hole 4-1 and an external circulating water outlet hole 4-2; the external circulating water inlet hole 4-1 is opened at the upper part of the outer tank shell 1, and is connected to the outlet of the temperature-controlled water tank through a pipeline, and the external circulating water outlet hole 4-2 is opened at the lower part of the outer tank shell 1, and is connected to the inlet of the temperature-controlled water tank through a pipeline; the liquid inlet pipeline, the liquid outlet pipeline and the air inlet pipeline are all immersed in the temperature-controlled water in the temperature-controlled chamber, so that the oxygen-containing nutrient solution and the oxygen-containing gas are preheated or precooled by the temperature-controlled water before entering the culture chamber and the in vitro intestinal sample.

[0076] Preferably, the isolated double tank further comprises an intracavitary perfusion inner tank inlet hole 5-2, an intracavitary perfusion inner tank outlet hole 5-3, a mesenteric vascular perfusion inner tank inlet hole 6-2, and a mesenteric vascular perfusion inner tank outlet hole 6-3, which are provided on the inner tank shell 2. The intracavitary perfusion inner tank inlet hole 5-2 serves as the outlet of the intracavitary perfusion inlet pipeline, the intracavitary perfusion inner tank outlet hole 5-3 serves as the inlet of the intracavitary perfusion outlet pipeline, the mesenteric vascular perfusion inner tank inlet hole 6-2 serves as the outlet of the mesenteric vascular perfusion inlet pipeline, and the mesenteric vascular perfusion inner tank outlet hole 6-3 serves as the inlet of the mesenteric vascular perfusion outlet pipeline. Taking the length direction of the isolated intestinal sample as the first direction, the intraluminal perfusion inner tank liquid inlet hole 5-2 and the intraluminal perfusion inner tank liquid outlet hole 5-3 are arranged relative to each other in the first direction, and the line between the two is parallel to the first direction; the mesenteric vascular perfusion inner tank liquid inlet hole 6-2 and the mesenteric vascular perfusion inner tank liquid outlet hole 6-3 are arranged relative to each other in the first direction, and the line between the two is parallel to the first direction.

[0077] Furthermore, the isolated double tank also includes an intracavitary perfusion outer tank inlet 5-1, an intracavitary perfusion outer tank outlet 5-4, a mesenteric vascular perfusion outer tank inlet 6-1, and a mesenteric vascular perfusion outer tank outlet 6-4, which are provided on the outer tank shell 1. The intracavitary perfusion outer tank inlet 5-1 and the intracavitary perfusion inner tank inlet 5-2 are connected by a pipeline to form an intracavitary perfusion inlet pipeline. The intracavitary perfusion inner tank outlet 5-3 and the intracavitary perfusion outer tank outlet 5-4 are connected by a pipeline to form an intracavitary perfusion outlet pipeline. The mesenteric vascular perfusion outer tank inlet 6-1 and the mesenteric vascular perfusion inner tank inlet 6-2 are connected by a pipeline to form a mesenteric vascular perfusion inlet pipeline. The mesenteric vascular perfusion inner trough outlet hole 6-3 and the mesenteric vascular perfusion outer trough outlet hole 6-4 are connected by a pipeline to form the mesenteric vascular perfusion outlet pipeline. The inlet of the intraluminal perfusion inlet pipeline is the intraluminal perfusion outer trough inlet hole 5-1, and the outlet is the intraluminal perfusion inner trough inlet hole 5-2. The inlet of the intraluminal perfusion outlet pipeline is the intraluminal perfusion inner trough outlet hole 5-3, and the outlet is the intraluminal perfusion outer trough outlet hole 5-4. The inlet of the mesenteric vascular perfusion inlet pipeline is the mesenteric vascular perfusion outer trough inlet hole 6-1, and the outlet is the mesenteric vascular perfusion inner trough inlet hole 6-2. The inlet of the mesenteric vascular perfusion outlet pipeline is the mesenteric vascular perfusion inner trough outlet hole 6-3, and the outlet is the mesenteric vascular perfusion outer trough outlet hole 6-4.

[0078] Furthermore, the isolated double tank also includes an outer inlet hole 7-1 for the inner tank nutrient solution and an outer inlet hole 9-2 for the inner tank nutrient solution, both provided on the outer tank shell 1, as well as an inner inlet hole 7-2 for the inner tank nutrient solution and an inner outlet hole 9-1 for the inner tank nutrient solution, both provided on the inner tank shell 2. The outer inlet hole 7-1 for the inner tank nutrient solution and the inner inlet hole 7-2 for the inner tank nutrient solution are connected by a pipeline to form an inner tank inlet pipeline. The inlet of the inner tank inlet pipeline is the outer inlet hole 7-1 for the inner tank nutrient solution, and the outlet is the inner inlet hole 7-2 for the inner tank nutrient solution. The inner outlet hole 9-1 for the inner tank nutrient solution and the outer outlet hole 9-2 for the inner tank nutrient solution are connected by a pipeline to form an inner tank inlet pipeline. The inlet of the inner tank nutrient solution infusion outlet line is the inner tank nutrient solution infusion outlet port 9-1, and the outlet is the inner tank nutrient solution outfusion outlet port 9-2. The inner tank nutrient solution infusion inlet port 7-2 is located at the bottom of the inner tank housing 2 and is connected to the inlet of the multi-outlet liquid pipeline 7-3. The inner tank nutrient solution infusion outlet port 9-1 is located at the top of the inner tank housing 2.

[0079] Furthermore, the isolated double tank also includes an external air inlet 8-1 provided in the outer tank shell 1, and an internal air inlet 8-2 provided in the inner tank shell 2. The external air inlet 8-1 and the internal air inlet 8-2 are connected by a pipeline to form an air inlet pipeline. The inlet pipeline has an inlet at the external air inlet 8-1 and an outlet at the internal air inlet 8-2. The internal air inlet 8-2 is located at the bottom of the inner tank shell 2 and is connected to the inlet of the multi-outlet gas pipeline 8-3.

[0080] During the process of solution circulation and introduction of oxygen-containing gas, the oxygen-containing nutrient solution and oxygen-containing gas flow through the temperature control chamber before being transported into the inner tank structure and the isolated intestinal sample, and are preheated or precooled under the regulation of the circulating water temperature in the temperature control chamber.

[0081] Specifically, the outlets of the multi-outlet liquid pipeline 7-3 are evenly distributed at the bottom of the culture chamber. The outlets of the multi-outlet gas pipeline 8-3 are evenly distributed at the bottom of the culture chamber.

[0082] In a specific implementation, the isolated double tank is a transparent container, preferably made of transparent acrylic material.

[0083] Preferably, the mesenteric vascular perfusion inner tank inlet hole 6-2 is correspondingly opened above the intraluminal perfusion inner tank inlet hole 5-2, and the mesenteric vascular perfusion inner tank outlet hole 6-3 is correspondingly opened above the intraluminal perfusion inner tank outlet hole 5-3.

[0084] Preferably, the oxygen-containing gas is 95% oxygen and 5% carbon dioxide.

[0085] Preferably, the nutrient solution is Krebs buffer.

[0086] The gas and liquid flow transmission paths in the in vitro intestinal activity maintenance device of the present invention are specifically as follows:

[0087] Temperature-controlled water circulation system: After the temperature-controlled water flows out of the outlet of the temperature-controlled water tank, it enters the temperature-controlled chamber from the external circulation water inlet hole 4-1. After the temperature-controlled water in the temperature-controlled chamber flows out from the external circulation water outlet hole 4-2, it flows back to the temperature-controlled water tank from the inlet; the temperature environment of the temperature-controlled chamber in the external tank can maintain the temperature in the culture room, and at the same time preheat or precool the perfusion liquid in the liquid inlet channel and the oxygen-containing gas in the air inlet channel, thereby improving the stability of the device in controlling the tissue environment temperature.

[0088] A double-pass aeration oxygenation system with multiple aeration points: the oxygen-containing gas in the gas source 15 is transported to the liquid storage tank 18 through the first branch and enters the culture chamber through the second branch. The path of the second branch is: after the oxygen-containing gas is output from the gas source 15, it enters the air inlet pipeline through the external air inlet hole 8-1, is fully preheated or precooled in the temperature control room by the buffer bottle 13, enters the multi-outlet gas pipeline 8-3 from the internal air inlet hole 8-2, and finally is evenly aerated into the oxygen-containing nutrient solution in the culture chamber from each outlet of the multi-outlet gas pipeline 8-3.

[0089] Multi-channel tissue fluid perfusion system: To address the presence of mesenteric blood vessels after intestinal surgical resection, the nutrient solution in the liquid storage tank 18 is mixed with oxygen-containing gas and then multi-channel perfused into the intestine, the intestine (intestinal lumen), and the blood vessels through three perfusion branches. First perfusion branch: The oxygen-containing nutrient solution is driven by a first solution pump and enters the intraluminal perfusion inlet pipeline from the intraluminal perfusion outer tank inlet hole 5-1. After being preheated or precooled in a temperature-controlled chamber, it enters one end of the intestine 11 through the intraluminal perfusion inner tank inlet hole 5-2. After passing through the lumen of the intestine 11, it enters the intraluminal perfusion outlet pipeline from the other end through the intraluminal perfusion inner tank outlet hole 5-3 and flows into the waste liquid storage tank 20 from the intraluminal perfusion outer tank outlet hole 5-4. Second perfusion branch: Oxygenated nutrient solution, driven by a second solution pump, enters the mesenteric vascular perfusion inlet pipeline through the outer mesenteric vascular perfusion tank inlet port 6-1. After being preheated or precooled in the temperature control chamber, it enters one end of the mesenteric vascular perfusion tank through the inner mesenteric vascular perfusion tank inlet port 6-2. After passing through the mesenteric vascular perfusion tank, it enters the mesenteric vascular perfusion outlet pipeline from the other end through the inner mesenteric vascular perfusion tank outlet port 6-3. It then flows into the waste liquid storage tank 20 through the outer mesenteric vascular perfusion tank outlet port 6-4. Third perfusion branch: Oxygenated nutrient solution, driven by a third solution pump, enters the inner nutrient solution perfusion inlet pipeline through the outer nutrient solution perfusion tank inlet port 7-1. After being fully preheated or precooled in the temperature control chamber by buffer bottle 13, it enters the multi-outlet liquid pipeline 7-3 through the inner nutrient solution perfusion tank inlet port 7-2. Finally, it is evenly injected into the culture chamber from each outlet of the multi-outlet liquid pipeline 7-3. The nutrient solution in the culture chamber enters the inner tank perfusion outlet pipeline through inner tank nutrient solution perfusion inner outlet port 9-1 at the top of inner tank housing 2 and flows into waste liquid storage tank 20 through inner tank nutrient solution perfusion outlet port 9-2. This multi-channel tissue fluid perfusion system comprehensively improves oxygen supply capacity, prevents the formation of hypoxic cores and necrosis within thick tissues, and prolongs the maintenance of active intestinal tract.

[0090] Preferably, the temperature control device 17 is a refrigeration device or a heating device. It can not only maintain the temperature control of body temperature conditions, but also increase the low temperature or sub-normal temperature control capability, providing a basis for physiological testing under multiple environmental conditions and extending the storage time.

[0091] A second aspect of the present invention provides an application of the above-mentioned isolated intestinal activity maintenance device.

[0092] The in vitro intestinal activity maintenance device of the present invention can be used for maintaining the activity of in vitro intestinal samples and for physiological activity testing, wherein the physiological activity testing includes electrophysiological testing, muscle tension testing, microscopic observation under active conditions, controlled pressure perfusion, and intracavitary pressure testing.

[0093] The third aspect of the present invention provides an application method of an isolated intestinal activity maintaining device.

[0094] The application method of the present invention comprises the following steps:

[0095] S1) turning on the temperature control device 17 and the external circulation water pump so that the temperature of the nutrient solution in the liquid storage tank 18 and the temperature-controlled water in the temperature-controlled water tank both reach a preset fixed temperature;

[0096] S2) Turning on the gas source 15, the oxygen-containing gas is delivered to the liquid storage tank 18 and the culture chamber of the isolated double tank at the set flow rates of the two gas paths; the nutrient solution in the liquid storage tank 18 is mixed with the oxygen-containing gas to form an oxygen-containing nutrient solution;

[0097] S3) turning on the solution pump 19 between the inner tank perfusion inlet line and the liquid storage tank 18 to continuously pump the oxygenated nutrient solution into the culture chamber through the inner tank perfusion inlet line, and then perfusing the oxygenated nutrient solution into the culture chamber through the inner tank perfusion inlet line and the liquid outlet line;

[0098] S4) If the intestinal tract 11 of the isolated intestinal sample is an intestinal segment, the two ends of the intestinal tract 11 are respectively connected and fixed to the intraluminal perfusion inner tank liquid inlet hole 5-2 and the intraluminal perfusion inner tank liquid outlet hole 5-3, and the solution pump 19 between the intraluminal perfusion liquid inlet pipeline and the liquid storage tank 18 is turned on to continuously pump oxygenated nutrient solution into the intestinal tract 11 of the isolated intestinal sample through the intraluminal perfusion liquid inlet pipeline, and then the oxygenated nutrient solution is perfused into 11 through the intraluminal perfusion liquid inlet pipeline and the liquid outlet pipeline;

[0099] If the intestinal tract 11 of the isolated intestinal sample is an intestinal slice, the intestinal tract 11 is fixed to the silicone rubber base 10 at the bottom of the inner tank using a sample fixing needle 24, and oxygen-containing nutrient solution is perfused into the bottom of the intestinal tract (11) through the intraluminal perfusion liquid inlet and outlet pipes to prevent hypoxia at the bottom of the intestinal slice sample;

[0100] S5) connecting and fixing the two ends of the mesenteric blood vessels 12 of the isolated intestinal sample to the mesenteric blood vessel perfusion inner tank inlet hole 6-2 and the mesenteric blood vessel perfusion inner tank outlet hole 6-3, respectively, turning on the solution pump 19 between the mesenteric blood vessel perfusion inlet pipeline and the liquid storage tank 18, continuously pumping oxygenated nutrient solution into the mesenteric blood vessels 12 of the isolated intestinal sample through the mesenteric blood vessel perfusion inlet pipeline, and then perfusing the mesenteric blood vessels 12 with oxygenated nutrient solution through the mesenteric blood vessel perfusion inlet pipeline and the mesenteric blood vessel perfusion outlet pipeline;

[0101] S6) After preserving the isolated intestinal sample at a preset temperature or incubating it for a period of time, a physiological activity test is performed while maintaining oxygen-containing gas delivery, nutrient solution perfusion, and temperature-controlled water circulation.

[0102] Step S6 specifically includes: storing the isolated intestinal sample at a preset temperature of 3 to 5° C. or incubating it at a preset temperature of 36 to 38° C. for 15 to 60 minutes, and then performing a physiological activity test.

[0103] Specifically, the physiological activity test is an electrophysiological test, a muscle tension test, microscopic observation under active conditions, or a controlled pressure perfusion and intracavitary pressure test.

[0104] The electrophysiological test process is as follows: a portion of the intestinal wall tissue of the intestine 11 is extracted to the end of the glass tube suction electrode 21 by negative pressure, the glass tube suction electrode 21 is electrically connected to the electrophysiological test system 22 and the host computer 23, and the electrophysiological test signal is collected;

[0105] The muscle tension test process is as follows: one end of the intestine 11 is fixed to the silicone rubber base 10 at the bottom of the culture chamber, and the other end is connected to the force sensor 25, which is electrically connected to the host computer 23 to collect the muscle tension test signal;

[0106] The microscopic observation process is specifically as follows: a light source 27 is integrated below the isolated intestinal sample, a microscope is arranged above it, and the microscopic structure of the isolated intestinal sample is observed using the microscope;

[0107] The controlled pressure perfusion and intracavitary pressure test process is specifically as follows: connecting the intracavitary perfusion outlet pipeline and / or the mesenteric vascular perfusion outlet pipeline to the electronic water pressure gauge 28, electrically connecting the force sensor 25 to the host computer 23, and monitoring the pressure changes in the cavity and / or mesenteric blood vessels in real time.

[0108] Furthermore, the device includes a venous effluent reservoir, to which the mesenteric vascular perfusion effluent line can be connected. Multiple perfusions are established by individually cannulating and mechanically perfusing the mesenteric vessels. This improves intestinal activity maintenance by delivering oxygenated perfusate through the arterial cannula and collecting venous effluent.

[0109] Preferably, during microscopic observation, the tissue is incubated with a calcium-sensitive fluorescent dye in a solution containing a solubilizer, and after washing, placed at the bottom of a culture chamber, and calcium ion fluorescence imaging is performed while maintaining intestinal activity.

[0110] Preferably, the composition of Krebs solution is customized based on the physiological environment characteristics of the tissue and the application requirements. When physiological activity testing is required under active conditions, the Krebs buffer is Krebs-Henseleit solution, which contains 118.3mM NaCl, 4.7mM KCl, 1.2mM MgSO4, 1.2mM KH2PO4, 25mM NaHCO3, 11.1mM D-glucose, and 2.5mM CaCl2.

[0111] The specific embodiments of the present invention are as follows:

[0112] Example 1

[0113] This embodiment provides an isolated intestinal activity maintenance device.

[0114] In this embodiment, the three-dimensional schematic diagram of the isolated double slot is as follows: Figure 1 The isolated double groove is made of acrylic material by laser or cutting. Among them, the outer tank shell 1 includes 4 liquid inlets (external circulating water inlet 4-1, intracavitary perfusion outer tank inlet 5-1, mesenteric vascular perfusion outer tank inlet 6-1, inner tank nutrient solution perfusion outer liquid inlet 7-1), 3 liquid outlets (intracavitary perfusion outer tank outlet 5-4, mesenteric vascular perfusion outer tank outlet 6-4, inner tank nutrient solution perfusion outer liquid outlet 9-3) and 1 air inlet (external air inlet 8-1); the inner tank shell 2 includes 3 liquid inlets (intracavitary perfusion inner tank inlet 5-2, mesenteric vascular perfusion inner tank inlet 6-2, inner tank nutrient solution perfusion inner liquid inlet 7-2), 3 liquid outlets (mesenteric vascular perfusion inner tank outlet 6-3, intracavitary perfusion inner tank outlet 5-3, inner tank nutrient solution perfusion inner liquid outlet 9-1) and 1 air inlet (inner air inlet 8-2). These channels are all provided with internal thread structures and are connected to hose connection interfaces with external threads.

[0115] In this embodiment, corresponding holes on the outer tank shell 1 and the inner tank shell 2 are connected using a flexible pipe such as a rubber tube.

[0116] In this embodiment, interfaces for securing ex vivo intestinal samples are provided on the inner side (located on one side of the inner wall of the inner tank housing 2) of the intraluminal perfusion inner tank inlet 5-2, the mesenteric vascular perfusion inner tank inlet 6-2, the mesenteric vascular perfusion inner tank outlet 6-3, and the intraluminal perfusion inner tank outlet 5-3. The interfaces have cavities connected to the channels, and the outer diameters of the interfaces are set according to the average inner diameters of the intestine 11 or mesenteric vessels 12. During sample securing, the intestine 11 or mesenteric vessels 12 can be respectively placed on the outer sides of the corresponding interfaces and then secured using methods such as wrapping surgical thread.

[0117] In this embodiment, the intestinal interface adopts a plastic hose connector, and the mesenteric blood vessels adopt a hose such as a rubber tube.

[0118] In this embodiment, the cross-sectional diagram of the internal connection of the isolated double slots is as follows: Figure 2 As shown. The outer tank's liquid inlet is connected to the corresponding inner tank's liquid inlet; the outer tank's liquid outlet is connected to the corresponding inner tank's liquid outlet; and the outer tank's air inlet is connected to the corresponding inner tank's air inlet. To ensure that the incoming culture fluid and gas are fully preheated or precooled in the outer tank's water temperature environment, the channels for delivering nutrient solution and oxygen to the inner tank are each connected to a buffer bottle 13. To prevent backflow of culture fluid after the device is shut down, the channels for delivering nutrient solution and oxygen to the inner tank are each connected to a check valve 14. Once connected, the cover plate 3 and the temperature control chamber are sealed.

[0119] In addition, in order to allow the nutrient solution and oxygen pumped into the inner tank to diffuse quickly to the entire intestinal segment, the nutrient solution and gas introduced through the inner liquid inlet hole 7-2 and the outer air inlet hole 8-1 of the inner tank are expanded through multiple channels and filled with polydimethylsiloxane. The multi-channel hose is fixed to the bottom of the inner tank. After the polydimethylsiloxane is cured, the silicone rubber interface protrudes at different positions of the entire intestinal segment.

[0120] In this embodiment, the schematic diagram of the connection between the isolated double slot and other devices is as follows: Figure 3 As shown, a temperature control device 17 regulates the overall water temperature, connects the isolated dual tanks, forms a circulation system, and pre-cools or pre-heats the nutrient solution storage tank 18. The oxygenated nutrient solution in the storage tank is controlled by a peristaltic pump and injected into the isolated dual tanks. It undergoes temperature pre-treatment in the external circulating water environment before flowing into the inner tank structure and being injected into the inner tank, the intestinal tract, and the mesenteric blood vessels. A 95% O2 / 5% CO2 gas source 15 is connected to two gas mass flow meters 16, which inject gas into the inner tank and the storage tank under controlled flow conditions for oxygenation. The nutrient solution drawn from the inner tank is transported via a pipeline to the waste liquid storage tank 20, establishing a complete fluid control system.

[0121] For maintaining the activity of human intestinal tissue, Krebs-Henseleit solution was used as a buffer solution, whose components included 118.3 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, 11.1 mM D-glucose and 2.5 mM CaCl2.

[0122] The process of performing activity maintenance operation using the in vitro intestinal activity maintenance device of this embodiment is as follows:

[0123] (1) Turn on the temperature control device and the external circulation water pump to keep the entire device at the set fixed constant temperature;

[0124] (2) Turn on the oxygen supply device to deliver 95% O2 / 5% CO2 to the liquid storage tank and the inner tank of the isolated double tank structure;

[0125] (3) Turn on the solution pump 19 to pump Krebs-Henseleit buffer into the inner tank of the isolated double tank structure;

[0126] (4) Fix the isolated intestinal tube to the inlet and outlet pipes of the inner tank and pump in Krebs-Henseleit buffer;

[0127] (5) The mesenteric vessels of the isolated intestinal tube are fixed to the inlet and outlet pipes of the inner tank, and Krebs-Henseleit buffer is pumped into the pipes;

[0128] (6) After incubating the isolated intestinal tube at the set temperature for 30 minutes, subsequent testing and application are carried out.

[0129] In this embodiment, for long-term storage of isolated intestinal tissue, the temperature control device 17 is a refrigeration device and maintains the device temperature at 4° C. For physiological activity testing of isolated intestinal tissue, the temperature control device 17 is a heating device and maintains the device temperature at 37° C.

[0130] Example 2

[0131] In this embodiment, Figure 4 A schematic diagram of electrophysiological testing after maintaining intestinal activity is provided. A glass tube suction electrode 21 is used to extract intestinal tissue while maintaining a negative pressure in the glass tube while keeping it filled with buffer. The glass tube electrode is then connected to an electrophysiological testing system 22 and host computer software 23 for electrophysiological testing.

[0132] Example 3

[0133] In this embodiment, Figure 5 A schematic diagram of muscle tension testing after maintaining intestinal activity is provided. The intestinal segment is secured to the bottom of the inner tank using a pin. The flexible polydimethylsiloxane silicone rubber base 10 facilitates smooth insertion and fixation. The free intestinal segment is connected to a force sensor 25 with the aid of surgical sutures and then connected to the host computer software to conduct muscle tension testing.

[0134] Example 4

[0135] In this embodiment, Figure 6 A schematic diagram of microscopic observation of the isolated intestine while maintaining intestinal activity is given. In order to ensure the irradiation effect of the light source on the sample, this application requires an improvement to the isolated double-tank structure. While keeping the fluid system unchanged and using transparent acrylic materials, the bottom area corresponding to the intestinal sample is hollowed out and a light source 27 is integrated so that the light source can fully illuminate the intestinal sample. An optical microscope 26 is set at the top of the intestine and connected to the host computer software 23 to observe the microstructure. In order to observe the ion movement of intestinal nerves, the tissue is incubated with a calcium-sensitive fluorescent dye in a solution containing a solubilizer, washed, and placed at the bottom of the inner tank. Calcium ion fluorescence imaging is performed while maintaining intestinal activity.

[0136] Example 5

[0137] In this embodiment, Figure 7 A schematic diagram of performing controlled pressure perfusion and intraluminal pressure testing while maintaining intestinal activity is provided. Connecting the intestinal perfusion output to an electronic water pressure gauge 28 and host computer software 23 enables real-time monitoring of intestinal pressure changes.

[0138] Comparative Example

[0139] In this comparative example, Figure 8 A schematic diagram of a general method for maintaining intestinal tissue activity is provided. After securing the intestinal tube, a nutrient solution inlet 29 and a 95% O2 / 5% CO2 inlet 30 are located on one side of the nutrient solution tank. The entire device is placed in a water bath for heating, and Kerbs solution is connected to both ends of the intestinal tube for perfusion.

[0140] This shows that, because the environment for maintaining intestinal tissue activity relies heavily on the diffusion of nutrients and oxygen, the device and method described in the comparative example are effective for maintaining the activity of thin tissues such as the mouse intestine. However, in thick tissues such as the human or pig intestine, hypoxic cores are very likely to form, causing cell death and tissue necrosis.

[0141] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. An in vitro intestinal activity maintenance device, characterized by: The in vitro intestinal activity maintaining device comprises an isolated double tank, a liquid storage tank (18), a temperature-controlled water tank, an air source (15), a temperature control device (17) and a waste liquid storage tank (20); a temperature-controlled chamber and a culture cavity are provided in the isolated double tank, the culture cavity is located in the temperature-controlled chamber, a multi-outlet liquid pipeline (7-3) and a multi-outlet gas pipeline (8-3) are arranged at the bottom of the culture cavity, the culture cavity is used to place the in vitro intestinal sample, and three liquid inlet pipelines, three liquid outlet pipelines and one gas inlet pipeline are arranged in the temperature-controlled chamber, the three liquid inlet pipelines and the liquid outlet pipelines are in one-to-one correspondence, and are respectively used to perfuse nutrient solution into the cavity, the mesenteric blood vessels and the inner tank; the temperature-controlled water tank The temperature control chamber is connected to the temperature control chamber through a temperature control water circulation pipeline. The gas source (15) is connected to the inlet of the gas inlet pipeline. The outlet of the gas inlet pipeline is connected to the inlet of the multi-outlet gas pipeline (8-3). The gas source (15) is connected to the gas inlet of the liquid storage tank (18). The liquid outlet of the liquid storage tank (18) is respectively connected to the inlets of the three liquid inlet pipelines. The outlet of the inner tank perfusion liquid inlet pipeline is connected to the inlet of the multi-outlet liquid pipeline (7-3). The inlet of the waste liquid storage tank (20) is respectively connected to the outlets of the three liquid outlet pipelines. The temperature of the nutrient solution in the liquid storage tank (18) and the temperature control water in the temperature control water tank are both controlled by the temperature control device (17).

2. The isolated intestinal activity maintaining device according to claim 1, characterized in that: Also includes: A buffer bottle (13), wherein at least one of the liquid inlet pipeline and / or the air inlet pipeline is provided with a buffer bottle (13); A check valve (14), wherein at least one of the liquid inlet pipeline and / or the air inlet pipeline is provided with a check valve (14); A solution pump (19) is installed on the pipeline between the liquid outlet of the liquid storage tank (18) and the inlet of each liquid inlet pipeline, and a solution pump (19) is installed on the temperature-controlled water circulation pipeline as an external circulation water pump; A gas mass flow meter (16) is installed on the pipelines between the gas source (15) and the liquid storage tank (18) and between the gas source (15) and the air inlet pipeline.

3. The isolated intestinal activity maintaining device according to claim 1, characterized in that: The isolated double tank mainly consists of an outer tank shell (1), an inner tank shell (2), a cover plate (3) and a silicone rubber base (10). The inner tank shell (2) and the outer tank shell (1) both adopt a top open structure. The inner tank shell (2) is arranged inside the outer tank shell (1). The middle area between the outer tank shell (1) and the inner tank shell (2) serves as a temperature control chamber. A cover plate (3) is arranged above the temperature control chamber. The cover plate (3) is detachably and sealedly connected to the outer tank shell (1) and the inner tank shell (2). The cavity inside the inner tank shell (2) serves as a culture chamber. The silicone rubber base (10) is laid on the bottom of the culture chamber. Each branch outlet of the multi-outlet liquid pipeline (7-3) and the multi-outlet gas pipeline (8-3) extends vertically upward to the top surface of the silicone rubber base (10) and opens upward. The isolated intestinal sample is an isolated intestinal sample from a pig or a human, and the isolated intestinal sample includes an intestinal tract (11) and / or a mesenteric blood vessel (12), and the intestinal tract (11) is an intestinal segment or an intestinal piece.

4. The isolated intestinal activity maintaining device according to claim 3, characterized in that: The outer tank shell (1) of the isolated double tank is provided with a plurality of outer tank liquid inlet holes, outer air inlet holes and outer tank liquid outlet holes, and the inner tank shell (2) is provided with a plurality of inner tank liquid inlet holes, inner air inlet holes and inner tank liquid outlet holes. The outer tank liquid inlet holes are connected to the corresponding inner tank liquid inlet holes through pipelines, the outer tank liquid outlet holes are connected to the corresponding inner tank liquid outlet holes through pipelines, and the outer air inlet holes are connected to the corresponding inner air inlet holes through pipelines, thereby forming a liquid inlet pipeline, a liquid outlet pipeline and an air inlet pipeline; the holes all have an internal thread structure and are connected to the pipelines through threaded connections.

5. The isolated intestinal activity maintaining device according to claim 3 or 4, characterized in that: The isolated double tank further comprises an external circulating water inlet (4-1) and an external circulating water outlet (4-2); the external circulating water inlet (4-1) is provided at the upper portion of the outer tank shell (1) and is connected to the outlet of the temperature-controlled water tank via a pipeline, and the external circulating water outlet (4-2) is provided at the lower portion of the outer tank shell (1) and is connected to the inlet of the temperature-controlled water tank via a pipeline; the temperature-controlled water tank is placed on the temperature control table of the temperature control device (17), and a liquid storage tank (18) is placed in the temperature-controlled water tank; the liquid inlet pipeline, liquid outlet pipeline and air inlet pipeline are all immersed in the temperature-controlled water in the temperature-controlled chamber.

6. The isolated intestinal activity maintaining device according to claim 3 or 4, characterized in that: The isolated double tank further comprises an intracavity perfusion inner tank liquid inlet hole (5-2), an intracavity perfusion inner tank liquid outlet hole (5-3), a mesenteric vascular perfusion inner tank liquid inlet hole (6-2), and a mesenteric vascular perfusion inner tank liquid outlet hole (6-3) provided on the inner tank shell (2); the intracavity perfusion inner tank liquid inlet hole (5-2) serves as the outlet of the intracavity perfusion liquid inlet pipeline, the intracavity perfusion inner tank liquid outlet hole (5-3) serves as the inlet of the intracavity perfusion liquid outlet pipeline, and the mesenteric vascular perfusion inner tank liquid inlet hole (6-2) serves as the outlet of the mesenteric vascular perfusion liquid inlet pipeline. outlet, the mesenteric vascular perfusion inner tank outlet hole (6-3) serves as the inlet of the mesenteric vascular perfusion outlet pipeline; with the length direction of the isolated intestinal sample as the first direction, the intraluminal perfusion inner tank inlet hole (5-2) and the intraluminal perfusion inner tank outlet hole (5-3) are arranged relative to each other in the first direction, and the line between the two is parallel to the first direction; the mesenteric vascular perfusion inner tank inlet hole (6-2) and the mesenteric vascular perfusion inner tank outlet hole (6-3) are arranged relative to each other in the first direction, and the line between the two is parallel to the first direction.

7. Use of the isolated intestinal activity maintenance device according to any one of claims 1 to 6, characterized in that: Used for activity maintenance and physiological activity testing of isolated intestinal samples, wherein the physiological activity testing includes electrophysiological testing, muscle tension testing, microscopic observation under active conditions, controlled pressure perfusion and intraluminal pressure testing, wherein the isolated intestinal sample is an isolated intestinal sample from a pig or a human, wherein the isolated intestinal sample includes an intestinal tract (11) and / or mesenteric blood vessels (12), and wherein the intestinal tract (11) is an intestinal segment or an intestinal slice.

8. A method for using the isolated intestinal activity maintenance device according to any one of claims 1 to 6, characterized in that: The following steps are involved: S1) Turn on the temperature control device (17) and the external circulation water pump; S2) opening the gas source (15) to deliver oxygen-containing gas to the liquid storage tank (18) and the culture chamber of the isolated double tank respectively; S3) perfusing the culture chamber with oxygenated nutrient solution through the inner tank perfusion inlet and outlet pipes; S4) If the intestinal tract (11) of the isolated intestinal sample is an intestinal segment, the two ends of the intestinal tract (11) are respectively connected to the intraluminal perfusion inner tank liquid inlet hole (5-2) and the intraluminal perfusion inner tank liquid outlet hole (5-3) and fixed, and oxygen-containing nutrient solution is perfused into the intestinal tract (11) through the intraluminal perfusion liquid inlet pipe and the liquid outlet pipe; If the intestinal tract (11) of the isolated intestinal sample is an intestinal slice, the intestinal tract (11) is fixed to the bottom of the culture chamber using a sample fixing needle (24), and oxygen-containing nutrient solution is perfused into the bottom of the intestinal tract (11) through the intracavitary perfusion inlet and outlet pipes; S5) connecting and fixing the two ends of the mesenteric blood vessels (12) of the isolated intestinal sample to the mesenteric blood vessel perfusion inner tank inlet hole (6-2) and the mesenteric blood vessel perfusion inner tank outlet hole (6-3), and perfusing the mesenteric blood vessels (12) with oxygenated nutrient solution through the mesenteric blood vessel perfusion inlet pipe and the outlet pipe; S6) Storing the isolated intestinal sample at a preset temperature or incubating it at a preset temperature for a period of time before performing a physiological activity test.

9. The application method according to claim 8, characterized in that: The step S6 specifically includes: storing the isolated intestinal sample at a temperature of 3 to 5° C. or incubating it at a temperature of 36 to 38° C. for 15 to 60 minutes, and then performing a physiological activity test.

10. The application method according to claim 8, characterized in that: The physiological activity test is an electrophysiological test, a muscle tension test, a microscopic observation under active conditions, or a controlled pressure perfusion and intracavitary pressure test; The electrophysiological test process specifically comprises: extracting part of the intestinal wall tissue of the intestine (11) to the end of the glass tube suction electrode (21) by negative pressure, electrically connecting the glass tube suction electrode (21) to the electrophysiological test system (22) and the host computer (23), and collecting the electrophysiological test signal; The muscle tension test process is specifically as follows: fixing one end of the intestine (11) to the bottom of the inner tank, connecting the other end to the force sensor (25), electrically connecting the force sensor (25) to the host computer (23), and collecting the muscle tension test signal; The microscopic observation process is specifically as follows: a light source (27) is integrated below the isolated intestinal sample, a microscope is arranged above the sample, and the microscopic structure of the isolated intestinal sample is observed using the microscope; The process of controlled pressure perfusion and intracavitary pressure testing is specifically as follows: connecting the intracavitary perfusion outlet pipeline and / or the mesenteric blood vessel perfusion outlet pipeline to the electronic water pressure gauge (28), electrically connecting the force sensor (25) to the host computer (23), and monitoring the pressure changes in the cavity and / or mesenteric blood vessels in real time.

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